The clinical incidence of infections caused by drug-resistant Gram-negative bacteria has increased dramatically in recent decades, especially for hospital-acquired infections. These infections can be essentially untreatable, leading to significant morbidity and mortality, as well as a high financial burden. Antimicrobial peptides (AMP) that target the microbial membrane directly have long been a promising but unfulfilled treatment alternative for drug-resistant, Gram-negative bacterial infections. But known AMPs have one or more of the following limitations to clinical usefulness: low solubility, residual toxicity, loss of effectiveness in the presence of concentrated host cells, and rapid degradation in plasma. In the proposed work, we will identify membrane targeting AMPs that circumvent all of the roadblocks to clinically useful activity. In the R21 portion we will identify gain-of-functin AMPs against Gram negative bacteria using screening of a validated iterative combinatorial peptide library that was based on a known AMP as a design template. We will complete the screening assay in which microbicidal activity is measured in the presence of highly concentrated human erythrocytes to mimic concentrated host cells encountered by peptides in vivo. If the hits identified satisfy our stringent milestones, we will progress to the R33 phase, performing extensive characterization of the selected hits. With this novel approach we will decipher the fundamental principles of useful AMP activity, and identify clinically-useful antimicrobial peptides that (1) are less than 15 residues long, and are linear and compositionally simple; (2) have broad- spectrum sterilizing antimicrobial activity against drug-resistant Gram negative bacteria at microM peptide in plasma and in the presence of concentrated host cells; (3) have little residual toxicity to human cells at high peptide concentration; (4) are highly soluble, and (5) are chemically stable in blood and plasma. Peptides with these properties will allow us to successfully exploit the bacterial plasma membrane as a novel target for drug-resistant bacterial infections.